Use of dkk-1 protein in the cancer diagnosis

ABSTRACT

Use of DKK-1 protein or the nucleic acid sequence in preparation of cancer diagnostic agents or kits, method to detect liver cancer with the monoclonal antibody thereof, the kit comprising anti-DKK-1 antibody or protein specific nucleic acid probes, together with a label, and method to detecting specific DKK-1 protein expression are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. Ser. No. 12/268532 filed Nov.11, 2008, which is a continuation of PCT application No.PCT/CN2006/000382 filed Mar. 13, 2006, which applications areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to molecular biology, particularly geneticdiagnosis. Specifically, the present invention relates to the use ofDKK-1 protein in diagnosis of cancer.

BACKGROUND ART

In 1998, Glinka A. et. al. published a research article in Nature(Nature, 1998; 391(6665):357-362), which claims that they have found anew secretory protein in the research work of the embryo development ofXenopus laevis, which is called dickkopf-1 (dkk-1). Their researchconfirms that dkk-1 is the inhibitory factor of Wnt signaling pathwayand is “inducer” formed in the “head induction” during the embryonicdevelopment of Xenopus laevis. Later, in 1999, Fedi P., et. al. (J BiolChem, 1999; 274(27):19465-72) isolated human homologous gene of dkk-1from human leiomyosarcoma cell SK-LMS-1 and the corresponding cDNAlibrary by conditioned chromatogram and PCR. The mRNA transcript isabout 2 kb, encoding 266 amino acids. Since then, the scientists for thefirst time reveal the molecular mechanism of DKK-1 as Wnt signalingpathway inhibitor through near 5 years of research work.

During the research work of the function and action mechanism of DKK-1,the scientists also notice that DKK-1 is relevant to certain humandiseases, such as osteoporosis (Biochem Biophys Res Commun, 2004;318(1):259-264. N Engl J Med, 2002; 346(20):1513-1521), bone damagecaused by multiple myeloma (N Engl J Med, 2003; 349(26):2483-2494) andother human malignancy. For example, Mikheev A M et. al.(Carcinogenesis, 2004; 25(1):47-59) established 2 non-tumorigenicrevertant cell lines with human cervical cancer Hela cell line, andfound by cDNA chip that DKK-1 is highly expressed in the above 2non-tumorigenic revertant cell lines and that lack of DKK-1 expressionis necessary for Hela tumogenicity. Therefore, DKK-1 is deemed as acandidate cancer inhibitory gene. Furthermore, Wirths O et. al. (LabInvest, 2003; 83(3):429-434) use “suppression subtractive hybridizationapproach” to find that DKK-1 is highly expressed in childrenhepatoblastoma and Wilms' tumor. The result shows that 26 out of 32children hepatoblastomas highly express DKK-1 (26/32 , 81%), 5 out of 6Wilms' tumors highly express DKK-1 (5/6 , 83%). However, only 2 out of20 liver cancer patients highly express DKK-1 (2/20 ,10%), and 1 out of5 medulloblastoma cell lines highly express DKK-1 (1/5 ,20%). There isno DKK-1 expression in malignant gliomas and breast cancers.

Therefore, there is urgent need for the precise and specific diagnosisof particular cancers.

SUMMARY OF THE INVENTION

Therefore, the object of the present invention is to provide diagnosiskit for precise diagnosis of selected cancer, the use of the DKK-1diagnosis kit, and the methods of in vitro detection of the expressionamount thereof.

In one aspect of the present invention, it provides a cancer diagnosiskit, wherein the said cancer selected from liver cancer, lung cancer,breast cancer and glioma, comprising containers with anti-DKK-1 antibodytherein. In one preferred embodiment of the present aspect, theanti-DKK-1 antibody is conjugated to detectable moieties. In a morepreferred embodiment, the detectable moieties are selected fromchromophore, chemical luminescent group, fluorophore, or isotopes.

In the second aspect of the present invention, it provides a cancerdiagnosis kit, wherein the said cancer is selected from liver cancer,lung cancer, breast cancer and glioma, comprising containers with DKK-1protein specific nucleic acid probe therein. In a further preferredembodiment of the present aspect, the probe is conjugated to detectablemoieties. In a more preferred embodiment, the detectable moieties areselected from chromophore, chemical luminescent group, fluorophore, orisotopes.

In the third aspect of the present invention, it provides the use ofDKK-1 protein or the nucleic acid sequence thereof in preparation ofcancer diagnostic agent or kit, wherein the cancer selected from livercancer, lung cancer, breast cancer and glioma. In one preferredembodiment of the present aspect, the cancer diagnostic agent isanti-DKK-1 protein specific antibody or DKK-1 protein specific nucleicacid probe.

In the fourth aspect, it provides a method for in vitro detectingspecific DKK-1 protein expression, comprising:

reacting anti-DKK-1 protein specific antibody or DKK-1 specific nucleicacid probe with cell sample, with normal cell as control;

comparing the binding amount of the antibody or probe, wherein theincreased amount over the control indicates the cancer cell, the loweror equal amount indicates normal cell.

In one preferred embodiment of the present aspect, the binding amount ismeasured by detecting the detectable moiety conjugated to the probe orantibody.

In the fifth aspect, it provides a method for detecting liver cancer,comprising the following step:

a) administering to an animal the anti-DKK-1 monoclonal antibodyconjugated to radionuclide;

b) detecting the gathering of anti-DKK-1 monoclonal antibody in vivo;

c) the said gathering indicates the presence of liver cancer.

In one preferred embodiment, the radionuclide is ¹³³I. In anotherpreferred embodiment, the animal is human.

DESCRIPTION OF FIGURES

FIG. 1 indicates the electrophoresis map of cRNA labeled by biotin. 1-5are sample numbers.

FIG. 2 indicates the electrophoresis map of the fragmented cRNA labeledby biotin. 1-5 are sample numbers.

FIG. 3 indicates the Northern hybridization expression analysis of DKK-1gene in 12 liver cancer patients.

FIG. 4 indicates the analysis of DKK-1 gene expression in 16 normalhuman tissues. The numbers are respectively: 1. spleen, 2. thymus, 3.prostate; 4. testicle; 5. ovary; 6. small intestine; 7. large intestine;8.peripheral blood lymphocyte; 9. heart; 10. brain; 11. placenta; 12.lung; 13. liver; 14. muscle; 15. kidney; 16. pancreas.

FIG. 5 is the radioactive immunoassay result of mice, wherein (a) is 24hours' display result; (b) is 48 hours' display result; (c) is 96 hours'display result; (d) is 120 hours' display result.

FIG. 6 is ¹³¹I-DKK1 distributing profile. As shown in the figure,¹³¹I-DKK1 gathers in the tumor tissue as time passes, and reaches thepeak at 120 hour. ¹³¹I-DKK1 reaches the peak in mice at 96 hour.

DETAILED EMBODIMENT OF THE INVENTION

The inventors compare difference of the gene expression profile of theliver cancer and the corresponding liver tissue adjacent to the cancerby genechip technology, and discover that it is contrary to the resultof Wirths O et. al. (Lab Invest, 2003; 83(3):429-434) that DKK-1 onlyexpresses in a few liver cancer patients (2/20, 10%). In the livercancer patients analyzed and confirmed by the inventors, 7 out of 12have DKK-1 high expression in the liver cancer tissue (7/12 , 58%),obviously higher than the result reported by Wirths O for the livercancer. Therefore, using ELISA, it is for the first time that the DKK-1amount in the serum of liver cancer patients is tested, which indicatesthat it highly expresses and secretes DKK-1. Based on this, it can beused in clinic diagnosis and therapy of liver cancer.

As used herein, the term “DKK-1” is a new secretory protein found in theresearch of Xenopus laevis embryo development, which is nameddickkopf-1(dkk-1). The sequence of the protein can be found by NCBIaccession number NP 571078. The DKK-1 protein herein comprises thecomplete amino acid sequence, the secretory protein, the mutant, and thefunctional active fragments. It should be understood that when encodingthe same amino acid, the nucleotide replacement in the codon isacceptable. Further to be understood is that in case of conservativeamino acid replacement produced by nucleotide substitution, thereplacement of nucleotide is acceptable.

When the amino acid fragments of DKK-1 are obtained, encoding nucleicacid sequences can be constructed, and the specific probes can bedesigned according to the nucleotide sequence. The full-lengthnucleotide sequence or the fragment thereof can be produced by PCRamplification, recombination or artificial synthesis. For PCRamplification, primers can be designed by the disclosed nucleotidesequences, particularly the open reading frame sequence, usingcommercially available cDNA library or cDNA library prepared by thecommon technology known by the skilled in the art to amplify thesequence. When the sequence is long, 2 or more times of PCRamplification is needed, then to link the fragments produced each timein right sequence.

Once relevant sequence is obtained, recombination method can be used tomassive produce the sequence, which commonly relates to clone thesequence into a vector, transfer into cell, and then isolate from thepropagated host cells by common method to obtain the sequence.

Furthermore, artificial synthesis can be used to synthesize thesequence, especially when the fragment is relative short. Usuallyfragments with long sequences can be obtained by first synthesizingmultiple small fragments and then linking them.

At present, the DNA sequence encoding the protein (or the fragments andderivatives) of the present invention can be produced by chemicalsynthesis. Then, the said DNA sequence can be introduced into any knownDNA molecules (or vectors) and the cells in the art.

By common recombination DNA technology, the present polynucleotidesequence can be used to express or produce recombinant DKK-1polypeptide. It usually comprises the following steps:

(1) Transform or transduct the suitable host cell by the presentpolynucleotide (or mutant) encoding human DKK-1 polypeptide, orrecombinant expression vector comprising the said polynucleotide;

(2) Culturing host cell in suitable culture medium;

(3) Isolating and purifying the protein from the culture medium or thecells.

In the present invention, DKK-1 polynucleotide sequence can be insertedinto recombinant expression vectors. Generally any vectors and plasmidscan be used as long as they can replicate and be stable in the host. Oneimportant character of the expression vector is that it usuallycomprises the replicate origin, promoter, marker gene and translationcontrol element.

Methods well known by the skilled in the art can be used to constructthe expression vectors comprising DKK-1 coding DNA sequence and suitabletranscription/translation control signal. These methods include in vitrorecombinant DNA technology, DNA synthesis technology, in vivorecombination technology, etc. The said DNA sequence can be effectivelylinked to suitable promoter in the expression vector to guide mRNAsynthesis. Expression vectors also include ribosome binding site forstarting the translation and the transcription terminators.

Furthermore, expression vector preferably comprises one or moreselective marker genes to provide the phenotypes which can be used toselect the transformed host cell, for example, dihydrofolate reductase,neomycin resistance and green fluorescent protein (GFP) for eukaryote,or tetracycline or ampicillin resistance for E. coli.

The vectors comprising the above suitable DNA sequences and suitablepromoters or regulating sequences can be used to transform suitable hostcells to express proteins.

The host cells can be prokaryotic cells, for example bacteria cells; orlower eukaryotic cells, for example yeast cells; or higher eukaryoticcells, for example mammalian cells. The representative examples are E.coli, bacteria cells of Streptomyces; fungal cells for example yeast;plant cells, insect cells; animal cells.

Transforming host cells with recombinant DNA can perform with commontechnology well-known by the skilled in the art. When the host isprokaryotes for example E. coli, the competent cells that can absorb theDNA can be obtained after the logarithmic growth phase, processed byCaCl₂ method, and the steps used are known in the art. The other methodis to use MgCl₂. If necessary, transforming can also useelectroporation. When the hosts are eukaryotes, following DNAtransfection method can be selected: calcium phosphate coprecipitation,normal mechanical methods such as microinjection, electroporation,liposome packing, etc.

The obtained transformant can be cultured with common method to expressthe polypeptide encoded by the present genes. According to the host cellused, the medium used in the cultivation can be selected from all kindsof regular mediums. The cultivation can be done under conditionssuitable for the growth of the host cells. When the host cells grow intothe desired cell density, suitable methods can be used to induceselected promoter (for example by temperature shift or chemicalinduction) and the cell can be cultured for some time more.

The recombinant polypeptides from the above method can be expressed inthe cell, on the cell membrane, or secreted outside the cell. Ifnecessary, recombinant proteins can be isolated and purified by variousisolation methods utilizing its physical, chemical or other characters.These methods are well known by the skilled in the art. Examples ofthese method include, but not limited to, regular renaturation process,protein precipitator treatment (salt out), centrifugation, osmoticbreaking-up of the bacteria, ultrasonication, ultracentrifugation,molecular sieve chromatography (gel filtration), adsorptionchromatography, ion exchange chromatography, high performance liquidphase chromatography (HPLC) and other liquid phase chromatography, andthe combinations thereof.

After obtaining the nucleic acid sequence, specific nucleic acid probecan be designed according to the sequence. Methods for designing theprobes are common in the art, See Sambrook et. al., Molecular Cloning, ALaboratory Mannual, Second edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, 1989. The exemplary method for testing whether DKK-1protein or nucleic acid exists in the biological samples comprisesexamining the biological sample of the subject, exposing the saidbiological sample to labeled nucleic acid probes that can hybrid withDKK-1 mRNA or genomic DNA. The nucleic acid probes can be for examplehuman nucleic acid or part of it, for example of at least 15, 30, 50, or100 nucleotides and the nucleic acid probes that can sufficiently hybridwith DKK-1 mRNA or genomic DNA. Other probes used in the presentdiagnosis assay are as mentioned herein.

Nucleic acid probes contact with the labeled and amplified sequences.The probes preferably are linked to chromophore, but can also beradioactive labeled. In another embodiment, the probes are linked tobinding partner, for example antibody or biotin, or another bindingpartner with a detectable domain.

In the traditional methods, the detection can be realized by Southernblotting and hybridization with labeled probes. The technology ofSouthern blotting is well known by the skilled in the art (See Sambrooket. al., 1989). Common detections also include biochips, florescentimaging technology, and flow cytometry, etc.

In another expect, the present invention also includes the specificpolyclonal antibodies and monoclonal antibodies, particular monoclonalantibodies, against polypeptides encoded by DKK-1 DNA or the fragmentsthereof. Herein, “specific” means that the antibody can bind with DKK-1gene product or fragments thereof, preferably those antibodies that bindwith DKK-1 gene product or fragment but do not recognize and bind othernon-relevant antigen molecules. The present antibody can be prepared byany technologies known by the skilled in the art.

The present invention not only include the complete monoclonal orpolyclonal antibodies, but also the antibody fragments that have immuneactivity, for example Fab' or (Fab)₂ fragments, antibody heavy chain,antibody light chain, single chain Fv molecules modified by geneticengineering, or chimeric antibodies.

Anti-DKK-1 antibody can be used in immunohistochemistry to detect theDKK-1 protein in the biopsy samples.

The direct detection of DKK-1 in blood or urine sample can beobservation index for tumor assistant diagnosis and prognosis, and canalso be the basis of tumor early diagnosis.

Antibodies can be detected by ELISA, Western blotting, or linked withdetecting groups, which can be detected by chemiluminescence, andisotopic tracing.

The present invention also includes kit to perform any method describedherein. In one non-limiting example, the kit can include one or more ofthe reagents with suitable forms of containers. The kit can alsocomprise reagents and labels for RNA isolation and purification of theRNA in the amplified cells.

The components of the kit can be packed in aqueous medium or inlyophilized form. The suitable containers in the kit usually at leastinclude one vial, tube, flask, bottle, syringe or other containers,which can have one components, and preferably suitably parted. Whenthere is more than one component in the kit, the kit will usually havesecond, third and other additional containers, which can separately holdadditional components. However, different combination of the componentscan be included in one vial. The present invention usually includes onecontainer to hold the reactants, which is sealed for commercialdistribution. The said container can include die-cast or blow moldedplastic containers, in which the necessary vial can be kept.

The present invention is further described hereinafter with particularexamples. It should be understood that these examples are just used toexplain the invention, not to limit the scope of the invention. Theexperimental methods without particular conditions in the followingexamples usually use the regular conditions, for example those describedin Sambrook et. al., Molecular Cloning, A Laboratory Mannual, ColdSpring Harbor Laboratory Press, New York, 1989, or as suggested by theproducers.

Example 1 Collection of the Tumor Tissue and Non-Cancerous Liver TissueSample from Liver Cancer Patients

12 samples of the tumor tissues from human hepatocellular carcinomas (T)and non-cancerous liver tissues (N) were collected from the liver cancerpatients from Shanghai, Guangxi, Qidong Jiangsu, and Hangzhou in China.In these samples, 1 from Shanghai (D129), 2 from Guangxi (G65 and G319),4 from Qidong Jiangsu (Q130, Q135, Q142, Q162), and 5 from Hangzhou(HK114, HK120, HK121, HK164, HK165). After operation, the tissue sampleswere immediately frozen in liquid nitrogen, and then stored in −80° C.ultra low temperature refrigerator.

Example 2 Isolation of RNA

1) 1 g tissue sample was triturated in liquid nitrogen into powder, thenimmediately added into 10 ml TRIZOL (Invitrogen, Cat 15596-026) tohomogenate. Left under room temperature (RT) for 10-15 minutes.

2) 2 ml chloroform was added and shaked vigorously 15 s. Left under RTfor 2-3 mins, centrifugated at 10,000 g at 4° C. for 15 mins.

3) Supernatant was collected, into which the same volume of isopropanolwas added. Left under RT for 2-3 mins, and then centrifugated at 10,000g at 4° C. for 15 mins.

4) Supernatant was discarded, 6 ml 75% ethanol was added to rinse thepellet. Centrifugated at 10,000 g at 4° C. for 15 mins.

5) The RNA pellet was slightly dried and solved in DEPC water.

6) The above crude total RNA sample was purified by RNeasy Mini Kit(Qiagen, Cat 74104) following the purification steps provided by theinstruction of the said kit from Qiagen. The purified DNA was kept under−70° C. for next use.

Example 3 cDNA Chip Hybridization Assay

1) UV Quantification and Detection:

UV spectrophotometer was used to detect the amount of the RNA. 1Absorbance Unit (OD) at 260 nm equals to about 40 μg/ml RNA. Accordingto the OD at 260 nm and 280 nm, the purity of the RNA is detected. Theratio of OD_(260 nm)/OD_(280 nm) of rather pure RNA should be near 2.0(preferable ratio should be 1.9-2.1).

2) cDNA Synthesis and Purification:

Synthesis of the first chain cDNA. Total RNA 5 μg was added withRNAse-free water into total volume 20 μl. T7-(dT)₂₄ primer 1 μl (100pmol/μl) was added and incubated at 70° C. for 10 min, then placed onice for at least 2 min and centrifuged. Then 5× first chain cDNAsynthesis buffer 4 μl, DTT (0.1M) 2 μl and dNTP (10 mM) 1 μl were added,mixed thoroughly, and incubated at 42° C. for 2 min. Then SuperScript IIRT (200 u/μl) 1 μl (5-8 μg starting RNA) was added, mixed thoroughly andincubated at 42° C. for 1 hour.

Synthesis of the second chain cDNA. The above RT-PCR synthesized firstchain cDNA product was left on the ice, and the following agents areadded: RNAse-Free water 92 μl, 5× second chain cDNA synthesis buffer 30μl, DTT (10 mM) 3 μl, E. coli DNA ligase (10 u/μl) 1 μl, E. coli DNApolymerase (10 u/μl) 4 μl, E. coli RNase H (10 u/μl) 0.2 μl. Thereactants were mixed thoroughly and reacted at 16° C. for 2 hours. ThenT4 DNA polymerase 3.3 μl was added, mixed thoroughly, and reacted at 16°C. for 5 min. Then 10 μl EDTA (0.5M) was added, mixed thoroughly to endthe reaction. The product was stored at −20° C.

cDNA purification. The above cDNA was purifed by Eppendorf PLG (PhaseLock Gel), PLG tubes were centrifuged at 12,000 g for 30 s.Phenol:Chloroform:isopropanol (25:24:1) was added at 1:1 into the cDNAreaction product, shaken vigorously. All the liquid was transferred intoPLG tubes without shake, centrifuged at 12,000 g for 2 minat RT. Thesupernatant was aspired into a new centrifuge tube, and 0.5 volume ofammonium acetate (7.5M, pH8.0) and 2.5 volume of pre-cooled absolutealcohol were added. The mixture was shaken thoroughly and centrifuged at12,000 g, RT for 20 min. Supernatant was discarded, and 75% ethanol 500μl was added, then centrifuged at 12,000 RT for 5 min. Again the cDNApellet was washed with 75% ethanol once, the liquid in the centrifugetube was discarded, air dry, and the pellet was solved in RNAse-freewater.

3) Synthesis of the Biotin-labeled cRNA:

Biotin-labeled cRNA was prepared by Enzo® BioArray™ HighYield™ RNAtranscript label kit (Enzo life sciences, INC). Above cDNA 5 μl wasadded with RNase-free water to 22 μl, and the following were added:10×HY reaction buffer (Tube No. 1) 4 μl, 10× biotin-labeled nucleotide(Tube No. 2) 4 μl, 10×DTT (Tube no. 3) 4 μl, 10× RNase inhibitor mixedsolution (Tube No. 4) 4 μl, 20×T7 polymerase (Tube No. 5) 2 μl. Themixture was mixed thoroughly, slightly centrifuged, and incubated at 37°C. for 4.5 h. Every 35 min, the mixture was centrifuged at 600 rpm for10 s. The synthesis product can be stored at −20° C. or directly used inthe next purification step.

Purification of cRNA. cRNA was purified with Qiagen kit. The method wassubstantially the same as for the total RNA (see steps 2-6).

Quantification and detection of cRNA. The cRNA concentration and theratio of OD260/OD280 were detected by UV spectrophotometer, and thequality of cRNA was assayed by denatured gel. 2 μg cRNA waselectrophoresized on 1.2% formaldehyde denatured gel, the purified cRNAvisualized as diffuse strip (FIG. 1).

Fragmentation of cDNA. cRNA 30 μg was added with 5× fragmentation buffer12 μl and RNAse-free water to 60 μl. Mixed thoroughly, incubated at 94°C. for 35 min, then placed on ice.

Detection of fragmented cRNA: 2 μg cRNA was electrophoresized on 1.2%formaldehyde denatured agarose gel, the visualized fragments of cRNAwere about 35-200 bp (FIG. 2).

4) Chip Hybridization:

The hybrid buffer was formulated. According to the chip type, suitableamount of hybrid buffer was formulated according to the following table.One sample chip needs 250 μl hybrid buffer, and one assay chip needs 90μl hybrid buffer. The formulation is provided in Table 1.

TABLE 1 The formulation of chip hybrid buffer Formulation 600 μl Finalconc. 1. Fragmented 60 μl 0.05 μg/μl cRNA (0.5 mg/ml) 2. Oligo B2control (3 nM) 10 μl 50 pM 3. 20x eukaryotic 30 μl 1x hybridizationcontrol 4. Salmon sperm 6.46 μl 0.1 mg/ml (9.3 mg/ml) 5. Acetylated BSA15 μl 0.5 mg/ml (20 mg/ml) 6. 2x hybrid buffer 300 μl 1x 7. RNase-freewater 178.6 μl

According to the chip hybridization method provided by Affymetrix, theassay chips were first hybridized, washed and stained, and analyzed.Then, sample chips were hybridized and assayed according to the resultsof the assay chips. The procedure of the chip hybrid assay was simplydescribed as follows with step 1 and 2 performed simultaneously untilstep 3 to finish.

Step 1: Pre-hybridization of the chips. The chips were taken out andbalanced to RT. 1× hybrid buffer was added, and then pre-hybridized at45° C., 60 rpm for 10 min.

Step 2. Preparation of the hybrid buffer. Hybrid buffer was mixedthoroughly and incubated at 99° C. for 5 min. Then it was shifted to 45°C. and incubated for 5 min. Then the mixture was centrifuged at maximumspeed for 5 min.

Step 3. Hybridization of the chips. 1× hybrid buffer was aspired fromthe chips. The hybrid buffer was added into the chips, then hybridizedat 45° C. 60 rom for 16 h. After hybridization, the hybrid buffer in thechips was aspired, then Wash A was added for the rinse and stainprocess. 5) Elution of the Chips.

Elution program was run on Eluting workstation according to the chiptype and the Chip elution method provided by Affymetrix.

6) Scanning of the Chips.

According to the chip scan method provided by Affymetrix, chip wasscanned on the scanner.

The human whole genome expressing chip of Affymetrix (Affymetrix,GeneChip® human genome U133 plus 2.0 arrays) includes 4.7×10⁴ genetranscripts and the splice variants of about 3.85×10⁴ genes of the humancomplete genome. The said chip was used to analyze the gene expressionspectrum of the cancer tissue and non-cancerous liver tissue of thehuman liver cancer subjects, and it was found that DKK-1 gene was highlyexpressed in liver cancer, the expression in liver cancer tissue wasabout 30 times higher than non-cancerous liver tissue.

Example 4 Northern Hybridization

1) Preparation of Northern Film

Preparation. Electrophoresis chamber, gel plate and comb were dipped in3% oxydol for more than 15 min, then rinsed clean by autoclaved DEPCprocessed water. Graduate and conical flask were dipped in DEPC waterover night, 180° C. parched for 8 h. 10×MOPS formaldehyde gelelectrophoresis buffer (Huashun, Cat W67) was used to formulate 1×formaldehyde gel electrophoresis buffer 1000 ml: 10×MOPS formaldehydegel electrophoresis buffer 100 ml, 37% formaldehyde 20 ml, andRnase-free water 880 ml. Formulation of 5×RNA load buffer: 80 μl 500 mMEDTA (pH8.0), 720 μl 37% formaldehyde, 2 ml 100% glycerol. 3084 μlformamide, and 4 ml 10×MOPS formaldehyde gel electrophoresis buffer. Anamount of bromophenol blue was added, and RNAse-free water was added tomake the volume up to 10 ml. The preparation of 1% formaldehyde geldenatured gen: 1 g agarose (GIBCO BRL, Cat 15510-027) was weighted andRNase-free water 90 ml was added. The agarose was microwave melted andthen 1.8 ml 37% formaldehyde, 10 ml 10×MOPS formaldehyde gelelectrophoresis buffer was added, thoroughly mixed and then filled intogel. Before the electrophoresis, the gel was hold in 1× formaldehyde gelelectrophoresis buffer to balance at least 30 min. The denaturation ofRNA samples: From each sample, total RNA 10 μg was taken, and 1 volumeof 5×RNA load buffer was added for every 4 volumes of sample. Themixture was mixed thoroughly and then incubated at 65° C. for 10 min,and immediately put on ice.

The RNA sample after electrophoresis, transfer and denaturation was onformaldehyde gel denaturation electrophoresis for 4 h, using up-runcapillary method to transfer the RNA on the gel to the nylon film (S&S,Cat 99J071). Load of 500 g was proposed, and the transfer time was 18-24h. The film was taken out, and rinsed in Milli Q water for severalminutes. The film was dried at 37° C., baked at 80° C. for 1.5 h, toimmobilize the RNA on the nylon film.

2) Labeling and Purification of DNA Probes:

Preparation of the probes. DKK-1 gene was PCR amplified. Specific primerincluding its coding region was designed according to cDNA sequence ofhuman DKK-1 gene on NCBI website (using primer designing softwareprimer3.cgi v0.2a). Forward primer 5′GACCCAGGCTTGCAAAGTGACGGT3′ andreverse primer 5′AGGAGTTCACTGCATTTGGATAGCTGG3′. DKK-1 was amplifiedusing human placenta cDNA (BD Clontech) as template. PCR kit was BDAdvantae™2 PCR kit (Cat 639206). The reaction system is as follows: 10×reaction buffer 1.25 μl, forward and reverse primers (10 μM) each 1 μl,human placenta cDNA templates 1 μl, Advantage 2 Polymerase 0.5 μl andsterilized water 7.75 μl. Total reaction volume was 12.5 μl. Temperaturecondition was: 94° C. 30 sec , 72° C. 3 min, 5 cycles; 94° C. 30 sec,70° C. 30 sec, 72° C. 3 min, 5 cycles; 94° C. 30 sec, 68° C. 30 sec, 72°C. 3 min, 27 cycles. After reaction, 1% agarose gel electrophoresis wasused to recover and purify the specific bands. PCR products were linkedto TA clone, and then transformed into competent E. coli. TOP10^(F).White colonies were picked and inoculated into LB medium, 37° C.overnight. Plasmid DNA was extracted, identified by PCR method and EcoRI(Promega, Cat R6011) enzyme cleavage. The positive clones including theinserted fragments were sequenced. The correct positive clones weresequenced, the plasmids were extracted, and cleaved by EcoRI. DKK-1fragments were recovered by electrophoresis and stored at −20° C. foruse as probes.

Labeling the probes: DNA probes were labeled by radioactive [α-³²P]dCTP(Amersham Biosciences, Cat PB10205) by random primer method as follows:25 ng DKK-1 probe DNA was solved in RNase-free water (1-33 μl), left inboiled water for 5 min to denature the DNA, immediately left on ice for5 min and refrigerated centrifuged immediately. Following reagents wereadded into the above DNA sample sequentially: 10× labeling buffer(including the random primers) 5 μl, dNTPs (dATP, dTTP, dGTP each 2 μl)6 μl, [α-³²P]dCTP (3000 Ci/mmol, 50 μCi) 5 μl, DNA Polymerasel-klenowfragments (5 u) 1 μl. Incubated at 37° C. for 1 hr.

The labeled DNA probes were purified with QIAquick Nucleotide Removalkit (Qiagen, Cat 28304), according to the instruction provided by thecompany.

3) Hybridization:

The prepared films were wetted by Milli Q water, and put into 68° C.pre-heated hybridization buffer (BD Bioscience, Cat 636832) topre-hybridize more than 3 hours. Salmon sperm DNA was added to 100μg/ml.

The purified probes were heated in 95-100° C. water for 5 min, put onice bath immediately for 5 min, and added into hybrid tubes, thenhybridized at 68° C. for 18-24 h.

The films were washed to remove excess and non-specific hybridizedprobes. The solution for washing the films was Solution 1 (2×SSC, 0.05%SDS) and Solution 2 (0.5×SSC, 0.1% SDS).

4) Pressing the Film:

The films washed were closed in plastic films, then X ray films werepressed above and exposed at −70° C.

Northern hybridization assay was used to test the expression profiles ofthe cancer tissue and non-cancerous liver tissue of the 12 liver cancerpatients, and it was found that 7 patients only highly expressed DKK-1in cancer tissues, and the non-cancerous liver tissue in the samepatient did not express DKK-1 (FIG. 3). The load amount of each samplewas about 10 μg total RNA. Furthermore, in all the 16 normal tissuesanalyzed DKK-1 only expressed in placenta tissue, and in all the other15 normal adult tissues it did not express (FIG. 4). Each normal humantissue included about 2 μg polyA⁺ RNA.

Example 5 Elisa Assay for the DKK-1 Protein Amount in the PeripheralBlood of Liver Cancer Patients

Coating the 96-well Elisa plate. 50 μl, goat anti-human DKK-1 polyclonalantibodies (R&D systems, Inc., Cat AF1096, 100 ng/μl) were solved in4,950 μl PBS solution. Each well of the 96-well ELISA plate was added 50μl the above dilution and 4° C. overnight. The wells were rinsed by0.05% PBST solution 200 μl 3 times, each time 3 min.

Each well was added 100 μl PBS with 4% BSA (Sigma, Cat A3059-50G), blockat RT for 2 h, then rinsed with 200 μl 0.05% PBST 3 times, each time 3min.

10 μl serum was added into 90 μl PBS supplemented with 0.1% Tween and 1%BSA. Each sample was added duplicated, each well 50 μl. 2 μl recombinanthuman DKK-1 protein standard (R&D systems, Inc., Cat 1096-DK, 10 ng/μl)was added into 400 μl PBS supplemented with 0.1% BSA, and then dilutedby fold. 7 dilution degrees were set, each standard was added induplicate and each well was added 50 μl. The plate was incubated at RTfor 2 h, and then rinsed with 200 μl 0.05% PBS for 3 times, each time 3min.

20 μl biotin labeled goat anti-human DKK-1 antibody (R&D systems, Inc.,Cat. BAF1144 , 50 ng/μl) was diluted in 4,980 μl 0.1% BSA in TBS, eachwell was added 50 μl the above dilution. The plate was left at RT for 2h, then rinsed with 200 μl 0.05% PBS for 3 times, each time 3 min.

0.5 μl Horseradish peroxidase conjugated with streptavidin (Vectorlaboratories, Inc., Cat. SA-5004) was diluted into 5 ml buffer (10 mMphosphate, 0.15 M NaCl, 0.1% Tween 20, pH 7.8), and 50 μl above dilutionwas added into each well, incubated at RT for 30 min. The wells wererinsed by 200 μl 0.05% PBST for 3 times, each time 3 min.

100 μl OPD substrate solution [8 mg OPD (DakoCytomation, Cat. S2045)solved in 12 ml water and 5 μl H₂O₂] was added into each well todevelop, incubated RT for 30 min.

100 μl 0.5M H₂SO₄ terminate buffer was added into each well, and thewells without standards were used as blank control. The OD of each wellwere tested at 490 nm.

Log-log standard curve was plotted according to the standards (logODversus logCONCENTRATION), and then DKK-1 amount in each serum sample wascalculated.

ELISA results of the serum DKK-1 of liver cancer patients as follow: Theaverage value of DKK-1 in 34 normal human serums is about 3.61 μg/L(with the maximum of 8.21 μg/L). Average value in 14 cirrhosis patientsis about 2.93 μg/L (with the maximum of 10.73 μg/L). The average valueof 128 liver cancer patients is about 4.85 μg/L [wherein 10 are 10μg/L<DKK-1<20 μg/L; 1 DKK-1=144.4 μg/L. 11/128 make up to 8.59% of thetotal liver cancer patients tested. In the 11 patients with high DKK-1,2 are AFP negative liver cancer patients, and 1 is AFP<200 μg/L livercancer patient (AFP=60.15 μg/L), making up to 27.3% (3/11)].

Example 6 ELISA Assay and Analysis of DKK-1 Secreted by Multiple Kindsof in Vitro Cultivated Human Tumor Cells

35 mm petri-dish was used to anabiosis the cells. Cell was passed into35 mm dish when growing into 90% confluence. About 3×10⁵ cells and lmlmedium were added into each dish. 24 h later cell culture supernatantwas collected.

The coating conditions and method of the 96-well ELISA plate werecompletely the same as in Example 5. 20 μl cell culture supernatant wasadded into 80 μl PBS supplemented with 0.1% Tween and 1% BSA. Eachsample was added in duplicate, each well 50 μl. The rest experiment wasoperated completely according to Example 5.

In the control of calf serum complete medium and fetus bovine serumcomplete medium, the concentration of DKK-1 protein was substantially 0(Table 2). In the culture supernatants of control murine fibroblastNIH3T3 and murine normal liver fibroid cell HSC-T6, DKK-1 protein wasnot detected, either (Table 2). However, highly expressing DKK-1 proteinwas found in 8 human tumor cell or human embryonic kidney cell 293,wherein human cerebral glioma cell U251 is the highest (214.6 μg/ml)(Table 2).

TABLE 2 Assay of DKK-1 protein amount in the tumor cells culturesupernatant DKK-1 amount in the cell Cell type culture supernatant Calfserum complete medium control 0.0 Fetus bovine serum complete medium 1.8control Murine fibroblast NIH3T3 0.8 Murine normal liver fibroid cellHSC-T6 0.5 Large cell lung cancer A549 143.1 Ovarian cancer SKV03 15.5Gastric cancer SW-1900 51.0 Breast cancer MCF-7 69.5 human embryonickidney cell 293 51.9 Rat cerebral glioma C6 55.4 Human cerebral gliomaU251 214.6 Cervic cancer C33A 85.2 Cervic cancer HeLa 102.3 melanomaA375 74.8 Highly metastatic melanoma SCI-375 61.1 Low metastatichepatoma cell MHCC97-L 112.0 Highly metastatic hepatoma cell HCCLM3118.2 hepatoma cell HepG2 182.8 hepatoma cell Hep3B 58.7 hepatoma cellBEL-7402 79.1 hepatoma cell SMMC-7721 52.3 hepatoma cell HuH7 76.7

Example 7 Kit

A kit was prepared which comprised nucleic acid probes directed at DKK-1(prepared as in Example 4), PCR reaction buffers (for amplify DKK-1).According to standard protocol, cDNA from normal tissues and cancercells of 100 hepatoma patients were amplified. The detection resultsshowed that the expression amount of DKK-1 in hepatoma cells of the 100hepatoma patient was 100 times of the normal cell.

For the same reason, patients with lung cancer, breast cancer and gliomawere also detected. Same results with DKK-1 expression as 50-100 timeshigh were also obtained.

Example 8

A kit was used according to the method of Example 5, which comprisedspecific antibodies against DKK-1 (prepared as in Example 5) and ELISAreagents. According to standard protocol, ELISA assay was done fornormal tissues and cancer cells of 100 hepatoma patients. The detectionresults showed that the expression amount of DKK-1 in hepatoma cells ofthe 100 hepatoma patient was 100 times of the normal cell.

For the same reason, patients with lung cancer, breast cancer and gliomawere also detected. Same results with DKK-1 expression as 50-100 timeshigh were also obtained.

Example 9

Radioimmunoimaging and biological distribution research was done on nudemouse model with human hepatoma using DKK-1 specific monoclonalantibody.

1. Material and Method

1.1 11 4-6 weeks old about 20 g male BALB/C nude mice were grown inShanghai Cancer Institute. Anti-DKK-1 monoclonal antibody 500 μg/1 ml,mIgG1 antibody 1 mg/1 ml, available from R&D systems, Inc.

1.2 Cultivation of the Cells.

(1) Cell resuscitation: Frozen human hepatoma SMMC-7721 cell cryovialwas taken out, and fast cast into 37° C. bath to recover the temperatureand melted fast. (2) The cry vial was taken out of the bath and opened.Cell suspension was aspired with pipette, filled into centrifuge tubeand 5 ml culture medium (DMEM+10% calf serum) was added dropwise. Aftermixing, centrifuged at 1000 rpm for 5 min, and the supernatant wasremoved. Rinsed for 3 times. (3) After rinsing, the cells were dilutedwith culture medium, inoculated into culture flask, in which 5 mlculture medium was added, and then put in 37° C. CO₂ incubator. On nextday, the culture medium was changed and the incubation continued. (4)When the adherent cell were up to 80%, they were digested and dividedseveral flasks. The culture medium was aspired completely with pipette,and the culture was washed with PBS twice, and the PBS medium wasaspired. Several drops of trypsin were added, mixed thoroughly, and keptflat in 37° C. incubator for digesting 1 min. Under inverted microscopeit could be seen that the cells shrinked into round shape. 2 ml culturemedium was added to stop the digestion, and the cells were removed fromthe flask wall, and moved into clean tubes, 1000 rpm for 5 min.Supernatant was removed, 2 ml culture medium was added to resuspend thecell pellet, mixed thoroughly. (5) Cells were counted and average valuewas calculated.

1.3 Establishing of the Nude Mice Model with Human Hepatoma

SMMC-7721 cell were prepared with PBS into unicell suspension. 4 nudemice were each subcutaneously injected at the back SMMC-7721 cell0.2-1×10⁷/0.2 ml. When the tumor grew up to about diameter 0.8 cm, itwas removed by surgery. The tumor was immediately immersed into sterilephysiological saline, and cut into small parts of 2.0 mm diameter, thentransplanted subcutaneously in the right side of the back of the nudemouse with trocar. Such transplant were repeated several times (eachtime not more than 3 nude mice) to make the in vivo biologicalproperties of SMMC-7721 cell stable. 11 nude mice were selected totransplant subcutaneously in the right side of the back using aboveprocedure. After 5 weeks, the tumors grew into 1 cm in diameter for use.

1.4 Labeling anti-DKK-1 monoclonal antibody using modified chloramine Tmethod, using iodine [¹³¹I] to mark anti-DKK1 monoclonal antibody andIgG1, isolated and purified on Sephadex G50 column. The radioactivechemical purity of iodine [¹³¹I] was tested as 95%, radioactiveconcentration was 200 μCi/ml, radioactive specific activity was 5μCi/μg. The radioactive chemical purity of iodine [¹³¹I]-IgG1 was testedas 94%, radioactive concentration was 75 μCi/ml, and radioactivespecific activity was 1 μCi/μg

1.5 24h before assay, the mice were fed 1% KI water to block thyroid.The detailed experimental steps were as follows: (1) Assay group and theadministration protocol: nude mice were divided into A and B groups,each group having 6 and 5 nude mices. Group A were all injected at venacaudalis with 30 μCi iodine [¹³¹I]-DKK1 and group B were all injected atvena caudalis with 12 μCi iodine [¹³¹I]-IgG1. (2) Biologicaldistribution and visualization: SPET visualization. Mice wereanaesthetized by inhaling of ether after 24, 48, 96 and 120 hours afterinjection of labeled antibodies, fastened by adhesive plaster and ranSPECT. ADAC WeltesPlus double sensor SPECT was used, which equipped highenergy collimator, energy peak 364Kev±10% window width, matrix 64×64,magnification 2.02, preset collection count 100K. (3) After SPECT 24,48, 96, 120 h, mice were executed by pulling neck and tumor, blood,liver, lung and other organs were collected, weighted and counted forradioactivity.

2. Result

2.1 SPECT results: 1 in 6 experimental mice in Group I had positiveimage (See FIG. 5). Experimental group II did not have positive image.Group I evidently was advantageous than group II.

2.2 ¹³¹I-DKK1 distribution profile (See FIG. 6) showed that ¹³¹I-DKK1gathered in the tumor tissue as time passed, and reached the peak at 120h. ¹³¹I-IgG1 in mice reached peak at 96 h.

3. Discussion

The characters of nuclide tumor imaging, such as high specificity, highsensitivity, non-traumatic and systematic make it the preferredscreening examination for certain diseases and it is effective stagingtool before certain tumor therapies. The present experiment used in vivodistribution experiment with ¹³¹I labeled anti-DKK1 monoclonalantibodies, and demonstrated that after injection of anti-DKK1monoclonal antibody the tumor area has apparent radioactivity gathering,which indicates that anti-DKK1 monoclonal antibody can recognize thetumor and the normal tissue, and has high affinity to human hepatomacells.

All above-noted published references are incorporated herein byreference as individual reference is incorporated. Furthermore, itshould understand that the skilled in the art can modify and verify thepresent invention in light of the above disclosure of the presentinvention. Such equivalents are also encompassed in the scope of theclaims appended hereto.

1-3. (canceled)
 4. The method according to claim 13, wherein theradionuclide is ¹³³I.
 5. The method according to claim 14, wherein thesubject is human. 6-10. (canceled)
 11. The method of claim 14, whereinthe cancer is liver cancer.
 12. The method of claim 14, wherein theanti-DKK-1 specific antibody is a monoclonal antibody or a polyclonalantibody.
 13. The method of claim 14, wherein the anti-DKK-1 specificantibody is linked to a radionuclide.
 14. A method to detect cancer in asubject, wherein the cancer is selected from liver cancer, lung cancer,breast cancer and glioma, comprising: applying to a sample obtained froma subject an anti-DKK-1 specific antibody or a DKK-1 protein specificnucleic acid probe, wherein the anti-DKK-1 specific antibody binds toDKK-1 protein, wherein the DKK-1 protein specific nucleic acid probebinds to a nucleic acid sequence that encodes the DKK-1 protein, andwherein each of the DKK-1 protein and the nucleic acid sequence thatencodes the DKK-1 protein is a molecular marker for the cancer;measuring an amount of the DKK-1 protein or the nucleic acid sequencethat encodes the DKK-1 protein by measuring an amount of a first signalgenerated by a first labeling substance that is conjugated to theanti-DKK-1 specific antibody or an amount of a second signal generatedby a second labeling substance that is conjugated to the DKK-1 proteinspecific nucleic acid probe; and comparing the amount of the DKK-1protein or the nucleic acid sequence that encodes the DKK-1 protein inthe sample with an amount of the DKK-1 protein or the nucleic acidsequence that encodes the DKK-1 protein in a normal control, wherein anincrease in the amount of the DKK-1 protein or the nucleic acid sequencethat encodes the DKK-1 protein in the sample as compared to the amountof the DKK-1 protein or the nucleic acid sequence that encodes the DKK-1protein in the normal control indicates that the subject has the cancer.15. The method of claim 14, wherein the sample is a body fluid sample.16. The method of claim 15, wherein the body fluid sample is a bloodsample.